Isoform nell-1 peptide

ABSTRACT

This application is drawn to a method of using an isoform NELL-1 peptide, and compositions thereof for bone formation or for treating, preventing, or ameliorating osteoporosis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 13/256,931, filed Nov. 23, 2011, which is a U.S. National PhaseApplication under 35 U.S.C. 371(c) of International Application No.PCT/US10/28540, filed on Mar. 24, 2010, which in turn claims the benefitof U.S. Provisional Application No. 61/163,297 filed on Mar. 25, 2009,the teaching of which is incorporated herein by reference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with Government support under DE016107, awardedby the National Institutes of Health. The Government has certain rightsin the invention.

BACKGROUND OF THE INVENTION

There are many situations where bone formation and regeneration arerequired for treatment, e.g., alveolar bone grafting, craniofacialdistraction osteogenesis, spinal fusion, segmental long bone defects.

Defects in the process of bone formation and regeneration are linked tothe development of several human diseases and disorders, e.g.osteoporosis and osteogenesis imperfecta. Failure of the bone repair orcartilage repair mechanism is also associated with significantcomplications in clinical orthopedic practice, for example, fibrousnon-union following bone fracture, implant interface failures and largeallograft failures. The lives of many individuals would be improved bythe development of new therapies designed to stimulate and strengthenthe fracture repair process.

Any new technique to stimulate bone repair or cartilage repair would bea valuable tool in treating bone fractures. A significant portion offractured bones are still treated by casting, allowing naturalmechanisms to effect wound repair. Although there have been advances infracture treatment in recent years, including improved devices, thedevelopment of new processes to stimulate or complement the wound repairmechanisms would represent significant progress in this area.

The techniques of bone reconstruction, such as used to reconstructdefects occurring as a result of trauma, cancer surgery or errors indevelopment, would be improved by new methods to promote bone repair.Reconstructive methods currently employed, such as using autologous bonegrafts or bone grafts with attached soft tissue and blood vessels, areassociated with significant drawbacks of both cost and difficulty. Forexample, harvesting a useful amount of autologous bone is not easilyachieved, and even autologous grafts often become infected or sufferfrom resorption.

Readily available and reliable bone graft material is essential for manyorthopedic surgeries. The current gold standard for bone graft materialis autologous bone. However associated donor site morbidity includingpain, gait disturbance, thigh paresthesia for iliac crest donor sites,infection, neurologic deficits, and hematomas for calvarial grafts makeautograft harvest less than ideal. Thus, there is a need for betterautograft alternatives.

Efforts to influence bone repair using bone stimulating proteins andpeptides, e.g., bone morphogenic proteins (BMPs), resulted in onlylimited success. While BMP2 is FDA approved and clinically successful asan osteoinductive biologic, there are significant reported side effectsincluding life-threatening cervical swelling. Therefore there is need todevelop improved and safer therapeutic approaches.

Cartilage is a type of dense connective tissue. It is composed ofchondrocytes which are dispersed in a firm gel-like matrix. Cartilage isavascular (contains no blood vessels) and nutrients are diffused throughthe matrix. Cartilage is found in the joints, the rib cage, the ear, thenose, the throat, and between intervertebral disks.

Cartilage can be damaged by wear, injury, or diseases. As agingprogresses, the water and protein content of the body's cartilagechanges. This change results in weaker, more fragile and thin cartilage.Osteoarthritis is a common condition of cartilage failure that can leadto limited range of motion, bone damage and invariably pain. Due to acombination of acute stress and chronic fatigue, osteoarthritis directlymanifests itself in a wearing away of the articulating surface and, inextreme cases, bone can be exposed in the joint. In another example,loss of the protective stabilizing meniscus leads to increased jointlaxity or abnormal motions that lead to joint instability. The excessivemotion and narrowed contact area promotes early arthritic changes.

Although numerous methods have been described for treatment of cartilageproblems, it is clear that many are artificial or mechanically basedsolutions that do not seek to recreate normal cartilage tissue biology.Therefore, there is a need for methods for stimulating cartilageformation and repair.

Efforts have been continuously made to find better or alternativeosteoinductive agents and therapeutic approaches in treating bonerelated and cartilage related conditions.

SUMMARY OF THE INVENTION

This invention provides an isoform Nell-1 (ISN-1) peptide and methods ofmaking the isoform Nell-1 peptide.

In various embodiments, this invention provides a composition or a bonegraft for enhancing the bone formation in a subject in which it isimplanted. In some embodiments, the composition contains a biocompatiblematrix and an ISN-1 peptide, a related agent, or combination thereof.The composition can further comprise LNell-1 protein, a related agent,or a combination thereof.

In some embodiments, the composition can be a pharmaceutical compositionwhich comprises a suitable carrier or excipient. In some embodiments,the pharmaceutical composition can be formulated into suitableformulation for suitable route of administration.

In some embodiments, the composition can be a bone graft which containsa biocompatible matrix and an ISN-1 protein, a related agent, a cellexpressing an ISN-1 protein, or a combination thereof. In someembodiments, the graft material is resorbable or biodegradeable. In someembodiments, the graft material can be synthetic or naturally occurring(e.g., allograft). The matrix can include a biodegradable polymer. Thematrix can be impregnated with an ISN-1 protein or a related agent, acell expressing an ISN-1 protein or a related agent, or a combinationthereof. The bone graft material can further comprise LNell-1 protein ora related agent, a cell expressing LNell-1 protein or a related agent,or a combination thereof.

In various embodiments, this invention provides a method of increasingbone formation or regeneration. The method can be used for the repair ofbone fractures. The method comprises increasing concentration of anISN-1 gene product at or near the fracture site. In some embodiments,the method comprises introducing an osteogenic cell or bone precursorcell that over expresses ISN-1 into the fracture site. In someembodiments, the method comprises increasing the expression of ISN-1gene product in an osteogenic cell or bone precursor cell at or near thesite of the bone fracture.

In some embodiments, the fracture site is contacted with an ISN-1protein or a pharmaceutical composition thereof. The fracture site canbe contacted with LNell-1 protein in addition to the ISN-1 protein.

In various embodiments, this invention provides a method of treatingosteoporosis using an ISN-1, a related agent, or a composition thereof.

In various embodiments, this invention provides a method for inducingcartilage formation or repair using an ISN-1, a related agent, or acomposition thereof. The composition can include an ISN-1 or relatedagent, and optionally at least one other active agent, cells, andbiocompatible material implanted for the purpose of cartilage repair(i.e., hyaline cartilage, elastic cartilage, or fibrocartilage).

In various embodiments, the use of ISN-1 can be combined with the use ofLNell-1.

In various embodiments, this invention provides a method of expressing afunctional ISN-1 peptide in a cell, said method comprising providing anucleic acid construct including at least a nucleic acid encoding atleast an ISN-1 peptide in frame with a nucleic acid encoding a secretorysignal peptide; transfecting a cell with said nucleic acid construct;culturing said cell under conditions that permit expression of the ISN-1peptide; optionally collecting ISN-1 peptide secreted from the cellline; optionally substantially purifying the ISN-1 peptide; andoptionally testing the activity of the ISN-1 peptide to induce boneformation.

Related cell line and nucleic acid construct for expressing ISN-1 arealso provided.

In the aforementioned embodiments, the ISN-1 protein can be SNell-1protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows schematic comparison of TSP-1 with LNell-1 and SNell-1proteins.

FIG. 2 shows LNell-1 and SNell-1 expression after Osx and Runx2transfection in NMCCs. Mouse primary cells were transfected with control(Con), Osx and Runx2 plasmids. 48 hours after transfection, RNA sampleswere extracted for RT-PCR and medium collected for immunoprecipitation(IP) assay using Nell-1 C-terminal Ab crosslinked with protein G beads.(A) RT-PCR results after using LNell-1 or SNell-1 primers. (B) IPresults using C-terminal Nell-1 antibody recognizing both LNell-1 andSNell-1.

FIG. 3 shows the effects of LNell-1 and SNell-1 on Runx2, Osx, Ocexpression and mineralization.

FIG. 4 shows LNell-1 and SNell-1 protein express patterns in mice heads.

FIG. 5 shows calvarial defect healing with SNell-1.

FIG. 6A shows sequence of SNell-1 protein [Homo sapiens] (SEQ ID NO:1).

FIG. 6B shows sequence of SNell-1 DNA (CDS) [homo sapiens] (SEQ IDNO:2).

FIG. 7A shows sequence of LNell-1 protein [Homo sapiens] (SEQ ID NO:3).

FIG. 7B shows sequence of LNell-1 DNA (CDS) [homo sapiens] (SEQ IDNO:4).

DETAILED DESCRIPTION

The present invention provides an isoform Nell-1 peptide (generallyreferred as ISN-1 herein). The previously discovered Nell-1 peptide of810 amino acids is referred to as LNell-1 herein. One exemplary ISN-1 isa short peptide referred to as SNell-1 herein.

Definition

As used herein, the term “ISN-1” refers to a Nell-1 peptide where theTSP1-N [N-terminal thrombospondin-1 (TSP-1)-like domain present inNell-1 peptide is removed so as to be a peptide retaining the functionof Nell-1 having a molecular weight about 63 kD. Physical advantages ofa peptide having a lower molecular weight include, e.g., enhancedefficiency of delivery into a cell, ease of upstream processdevelopment—more efficient cell synthesis or secretion into media, easeof downstream process development—more efficient separation,purification, folding, etc. Biological advantages include increasedosteogenic differentiation as evidenced by increased expression ofosteoblastic differentiation markers Runx2, Osx, and Oc (FIG. 3) andbone formation.

The term “osteogenic cells” refers to cells capable of mineralizing.Osteogenic cells include osteoblasts, osteoblast like cells, mesenchymalcells, fibroblast cells, fetal embryonic cells, stem cells, bone marrowcells, dura cells, chrondrocytes, and chondroblastic cells.

As used herein, the term “bone precursor cells” refers to the cells thatcan differentiate into osteoblasts upon exposure to a bone growth factorand deposit calcium into the extracellular matrix.

As used herein, the term “bone progenitor cells” refers to any or all ofthose cells that have the capacity to ultimately form, or contribute tothe formation of, new bone tissue. This includes various cells indifferent stages of differentiation, such as, for example, stem cells,bone marrow cells, fibroblast cells, vascular cells, osteoblast cells,chondroblast cells, osteoclast cells, and the like. Bone progenitorcells also include cells that have been isolated and manipulated invitro, e.g. subjected to stimulation with agents such as cytokines orgrowth factors or even genetically engineered cells. The particular typeor types of bone progenitor cells that are stimulated using the methodsand compositions of the invention are not important, so long as thecells are stimulated in such a way that they are activated and, in thecontext of in vivo embodiments, ultimately give rise to new bone tissue.

The term “osteochondroprogenitor” refers to any cell capable of formingcartilage, e.g., less differentiated osteogenic cells which are capableof mineralizing and/or forming cartilage. Osteochondroprogenitor cellsinclude osteoblasts, osteoblast like cells, mesenchymal cells,fibroblast cells, fetal embryonic cells, stem cells, bone marrow cells,dura cells, chrondrocytes, and chondroblastic cells.

The term “osteoporosis” refers to a heterogeneous group of disorderscharacterized by decreased bone mass and fractures. Clinically,osteoporosis is segregated into type I and type II. Type I osteoporosisoccurs predominantly in middle aged women and is associated withestrogen loss at the menopause, while osteoporosis type II is associatedwith advancing age.

The term “cartilage” refers to all forms of cartilage including, but notlimited to, hyaline, elastic, and fibrocartilage.

The term “nucleic acid” or “oligonucleotide” herein refers to at leasttwo nucleotides covalently linked together. A nucleic acid of thepresent invention is preferably single-stranded or double stranded andwill generally contain phosphodiester bonds, although in some cases, asoutlined below, nucleic acid analogs are included that can havealternate backbones, comprising, for example, phosphoramide,phosphorothioate, phosphorodithioate, O-methylphophoroamidite linkages,and peptide nucleic acid backbones and linkages. Other analog nucleicacids include those with positive backbones, non-ionic backbones, andnon-ribose backbones. Nucleic acids containing one or more carbocyclicsugars are also included within the definition of nucleic acids.Modifications of the ribose-phosphate backbone can be done to facilitatethe addition of additional moieties such as labels, or to increase thestability and half-life of such molecules in physiological environments.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers.

The term “cell adhesion molecules” refers collectively to laminins,fibronectin, vitronectin, vascular cell adhesion molecules (V-CAM) andintercellular adhesion molecules (I-CAM) and collagen.

The terms “carrier,” or “pharmaceutically acceptable carrier,” or“delivery vehicle,” or “vehicle” can be used interchangeably.

The terms “increasing”, “enhancing”, and “facilitating” may be usedinterchangeably.

As used herein, the term “Nell-1 peptide” can include a Nell-1 relatedagent. For example, a Nell-1 peptide related agent can include anypolypeptide with significant homology to a Nell-1 peptide or a fragmentthereof. Significant homology can be a homology of higher than about 50%homology to a Nell-1 peptide, e.g., higher than about 60% homology to aNell-1 peptide, higher than about 70% homology to a Nell-1 peptide, orhigher than about 80% homology to a Nell-1 peptide. Nell-1 peptide maybe referred simply as Nell peptide herein.

The Nell-1 peptides can be natural and/or recombinant Nell-1 peptideswith a non-mutated wild-type sequence or recombinant Nell-1 peptideswith a mutated wild-type sequence that still contains significanthomology to Nell-1 peptides. In addition, Nell-1 peptides can be derivedfrom, but not limited to, an organism such as human cells, bacteria,yeast, or insect or plant cells. In some embodiments, the term “Nell-1peptide” includes structural, functional or conformational equivalentsof Nell-1 peptide. As used herein, a structural equivalent of a Nell-1peptide refers to a protein or peptide including a structure equivalentor substantially similar to that of a Nell-1 peptide or of a functionaldomain of a Nell-1 peptide. A functional equivalent of a Nell-1 peptiderefers to a protein or peptide having a function equivalent orsubstantially similar to that of a Nell peptide or of a functionaldomain of a Nell-1 peptide. A conformational equivalent of a Nell-1peptide refers to a protein or peptide having a conformation equivalentor substantially similar to that of a Nell-1 peptide or of a functionaldomain of a Nell-1 peptide.

In some embodiments, the Nell-1 peptide described herein can be aderivative of the Nell-1 peptide. The term “derivative” as used herein,refers to any chemical or biological compounds or materials derived froma Nell-1 peptide, structural equivalents thereof, or conformationalequivalents thereof. For example, such a derivative can include anypro-drug form, PEGylated form, or any other form of a Nell peptide thatrenders the Nell-1 peptide more stable or to have a betterosteophilicity or lipophilicity. In some embodiments, the derivative canbe a Nell-1 peptide attached to poly(ethylene glycol), a poly(aminoacid), a hydrocarbyl short chain having C1-C20 carbons, or abiocompatible polymer. In some embodiments, the term “derivative” caninclude a Nell-1 peptide mimetics. Synthesis of mimetics of a peptide iswell documented in the art.

In some embodiments, the peptide derivative described herein includes aphysically or chemically modified Nell-1 peptide. Physically modifiedpeptide can be modification by, for example, modification by ionic forcesuch as forming an ionic pair with a counterion, modification byhydrogen bonding, modification by modulation of pH, modulation bysolvent selection, or modification by using different proteinfolding/unfolding procedures, which can involve selection offolding/unfolding temperature, pH, solvent, and duration at differentstage of folding/unfolding.

In some embodiments, the peptide derivative can include a chemicallymodified Nell-1 peptide. For example, a short hydrocarbon group(s) (e.g.methyl or ethyl) can be selectively attached to one or multiple sites onthe Nell-1 peptide molecule to modify the chemical and/or physicalproperties of the peptide. In some embodiments, a mono-, oligo- orpoly(ethylene glycol) (PEG) group(s) can be selectively attached to oneor multiple sites on the Nell-1 peptide molecule to modify the chemicaland/or physical properties of the peptide by commonly known proteinPEGylation procedures (see, e.g., Mok, H., et al., Mol. Ther.,11(1):66-79 (2005)).

In the same vein, isoform Nell-1 peptide can include an isoform Nell-1related agent or derivative. The above described principles areapplicable to the isoform Nell-1 peptide.

Isoform Nell-1 Peptides

Nell-1 peptide, an 810 amino acid peptide with a molecular weight of 90kD, has been found to have osteoinductive properties. Nell-1 peptide,methods of its expression and use in treating bone and cartilage relatedconditions have been described in U.S. Pat. Nos. 7,052,856 and7,544,486, U.S. application Ser. Nos. 11/392,294, 11/594,510,11/601,529, 11/713,366, 11/884,525, and 11/973,831.

The isoform Nell-1 in the present invention does not include the Nell-1described in the previous patents or patent applications identifiedabove.

Isoform Nell-1 (referred to as ISN-1 herein) is a Nell-1 peptide lackinga TSP1-N [N-terminal thrombospondin-1 (TSP-1)-like domain present inLNell-1. One exemplary ISN-1 is a short Nell-1 (SNell-1) of sequence ofSEQ ID NO:1.

Rat or mouse isoform Nell-1 peptides share ˜93% predicted amino acidhomology with human Nell-1. LNell-1 contains several highly conservedmotifs including a secretory signal peptide, an N-terminalthrombospondin-1 (TSP1-N)-like module (also described as laminin G-likedomain), five chordin-like cysteine-rich (CR) domains (also known as vonWillebrand factor type C domains) and six epidermal growth factor(EGF)-like domains. Rat LNell-1 is secreted into media as 400-kDaproteins that convert to 130-kDa proteins after prolonged denaturation.The 130-kDa monomers are assumed to associate into homotrimers viaeither the coiled-coil region or CR domains. The EGF-like domainsinteract with and are phosphorylated by protein kinase C (PKC) in a PKCisoform specific manner. (FIG. 1)

Human LNell-1 contains 810 amino acids with a molecular weight of 89.5kD (˜120 kD after post-translational modification). LNell-1 istranscribed from the proximal alternative promoter (AP-L).

SNell-1 was predicted to have 570 aa with a molecular weight of 62.5 kD.SNell-1 is transcribed from a novel alternative promoter (AP-S). BothAP-L and AP-S contain multiple functional regulatory elements forbinding Runx2 (e.g., OSE2) and Osx [e.g., specificity protein 1 (SP1)].

Runx2 promotes LNell-1 and SNell-1 mRNA/protein expression, while Osxinduces SNell-1 mRNA/protein, but suppresses LNell-1 mRNA expression,indicating that LNell-1 and SNell-1 have distinct roles at early andlate stages respectively of osteoblast differentiation. Moreover,LNell-1 can reciprocally downregulate Osx transcription, while SNell-1can upregulate both Runx2 and Osx transcripts and increases osteoblasticdifferentiation. Like LNell-1, SNell-1 is expressed during skeletalgrowth and demonstrates osteogenic potential in vitro and in vivo.

Both Nell-1 isoforms can be required during osteogenesis. LNell-1 andSNell-1 have distinct, non-overlapping functions during osteogenesis andchondrogenesis. SNell-1, which upregulates both Runx2 and Osx, isbelieved to be an even more potent osteoinductive agent than LNell-1.

SNell-1 peptide is of a sequence of SEQ ID NO:1. SNell-1 peptide isencoded by a DNA sequence of SEQ ID NO:2. LNell-1 peptide is of asequence of SEQ ID NO:3. LNell-1 peptide is encoded by a DNA sequence ofSEQ ID NO:4.

Method of Increasing Bone Formation and Regeneration

This invention provides a method of increasing bone formation andregeneration. The method can be used for bone fracture repair. Themethod is useful in a variety of contexts including, but are not limitedto, bone reconstruction of defects occurring as a result of trauma,cancer surgery or errors in development, treatment of osteogenesisimperfecta, treatment of osteoporosis, and the healing of major or minorbone fractures.

The method for bone fracture repair comprises increasing concentrationof an ISN-1 gene product at or near the fracture site. In someembodiments, the method comprises transfecting an osteogenic cell with avector that expresses ISN-1 protein or a related agent at or near thebone fracture site. In some embodiments, the method comprisesintroducing an osteogenic cell or bone precursor cell that overexpressesISN-1 into the fracture site.

In another approach to fracture repair, the fracture site is contactedwith an ISN-1 protein. In some embodiments, the fracture site iscontacted with LNell-1 in addition to an ISN-1 protein. The protein canbe produced by a cell (e.g. introduced by introduction of a celloverexpressing Nell-1 protein), or by administration of the proteinalone or in combination with a pharmacological excipient, or byadministration of a “naked DNA” vector capable of expressing Nell-1peptide or the ISN-1. The ISN-1 protein can be a component of a bonerepair/bone graft material and/or part of a prosthetic device.

In some embodiments, in a manner analogous to the use of bonemorphogenic proteins (e.g. BMP-1 through BMP-24), the ISN-1 can be usedto speed repair of bone fractures or to induce bone repair orreplacement under circumstances where natural healing is limited ornon-existent. In general, such methods involve increasing the amount ofa Nell-1 gene product at or near the fracture site in a bone. The ISN-1gene product concentration can be increased by one or more of a numberof methods. In one approach, cells at or near the bone fracture site areinduced to express elevated levels of ISN-1. This can be accomplished invivo, for example, by the use of modulators of Nell-1 expression, byaltering the ISN-1 promoter, or by transfecting the cell with aconstruct that expresses ISN-1. This also can be accomplished bymodifying such cells to overexpress Nell-1 ex vivo and then introducedback into the subject organism (e.g. at or near a fracture site).

In various embodiments, this invention provides a method of facilitatingbone formation or regeneration, the method comprising increasing theconcentration of an ISN-1 gene product in an osteogenic cell. The ISN-1gene product can be an ISN-1 peptide, a related agent, or a combinationthereof. In some embodiments, the osteogenic cell can be a matureosteoblast, osteoblast, a mesenchymal cell, a fibroblast cell, a fetalembryonic cell, a stem cell, a bone marrow cell, a dura cell, achrondrocyte, and a chondroblast.

In some embodiments, the increasing concentration of an ISN-1 geneproduct comprises transfecting an osteogenic cell with a vector thatexpresses an ISN-1 protein or a related agent. In some embodiments, theincreasing concentration of an ISN-1 gene product comprisesadministering to the bone fracture site with a composition comprising anISN-1 protein or a related agent. The composition can further comprise apharmaceutically acceptable carrier.

In various embodiments, the use of ISN-1 and LNell-1 can be combined. Itis expected that ISN-1 and LNell-1 can work in synergy and thecombination provides improvement to the existing approaches.

In various embodiments, the isoform Nell-1 can be SNell-1.

Method of Treating Osteoporosis

The use of Nell-1 for treating, preventing, and amelioratingosteoporosis has been described in U.S. application Ser. No. 11/713,366,the teaching of which is incorporated by reference herein.

In various embodiments, this invention provides a method of treating,preventing or ameliorating osteoporosis by administering to a bonetissue at a pre-selected site an effective amount of an ISN-1 or relatedagent.

In some embodiments, the method can further comprise applying to thepre-selected site a physical force to disperse the ISN-1 or relatedagent. In some embodiments, the physical force can be ultrasound.

In some embodiments, the administering step comprises: making anincision in the bone tissue at the pre-selected site, and delivering tothe bone tissue at the pre-selected site via the incision.

In some embodiments, the ISN-1 or related agent is formulated into aformulation suitable for a mode of delivery selected from percutaneousinjection through intact skin to a site, direct injection through asurgically opened site or a trauma site, surgical implantation,extravascular delivery, extravascular injection, extravascular catheterbased injection, intravascular delivery, intravascular injection,intravascular catheter based injections, intravenous delivery,intravenous injection, intravenous catheter based injections,intraarterial delivery, intraarterial injection, intraarterial catheterbased injections, intrathecal delivery, intrathecal injection,intrathecal catheter based injections, intraosseous delivery,intraosseous injection, catheter based injections, intracartilaginousdelivery, intracartilaginous injection, intracartilaginous catheterbased injections, intravesical delivery, intravesical injection,intravesical catheter based injection, delivery via a mechanical pumpwith a percutaneous or implantable catheter, catheter based delivery toan area or organ in the body, or delivery via expanded dispersionthrough a device that increases tissue penetration or wider tissuedistribution. In some embodiments, the device provides ultrasound,iontophoresis, heat or pressure.

In various embodiments, the use of ISN-1 and LNell-1 can be combined. Itis expected that ISN-1 and LNell-1 can work in synergy and thecombination provides improvements to the existing approaches. In variousembodiments, the isoform Nell-1 can be SNell-1.

Method of Inducing Cartilage Formation and Regeneration

The use of Nell-1 for inducing cartilage formation and regeneration hasbeen described in U.S. application Ser. No. 11/594,510, the teaching ofwhich is incorporated by reference herein.

In various embodiments, the present invention provides agents andmethods for inducing cartilage formation or repair using an ISN-1peptide or a related agent (collectively referred as “agent”). Thecomposition can include an ISN-1 peptide, a related agent, andoptionally at least one other active agent, cells, and biocompatiblematerial implanted for the purpose of cartilage repair (i.e., hyalinecartilage, elastic cartilage, or fibrocartilage).

In some embodiments, the present invention provides a composition thatcontains an effective amount of at least one agent for either directlyor indirectly promoting the generation of cartilage for treating,preventing or ameliorating a cartilage related medical condition. One ofthe agents for direct promotion of cartilage generation can be ISN-1peptides or ISN-1 based gene therapy or ISN-1 gene product enhancersapplied to chondrogenic cells such as, but not limited to,chondroblasts, chondrocytes, or chondroprogenitor cells, adult andembryonic stem cells, bone marrow cells, bone marrow stromal cells,mesenchymal cells, a fibroblast, or adipose derived cells. The agent forindirect promotion of cartilage generation (e.g., through inducingchondroblast/chondrocyte differentiation) can be, e.g., one of Nellpeptide, or agonists of Nell peptide receptors.

In some embodiments, the composition can include, e.g., one or moreinhibitors or antagonists of ISN-1 peptide receptors, high dose ISN-1peptides, or combinations thereof. Such a composition is effective forinhibition of chondrogenic differentiation by inhibiting potential orcommitted chondrogenic cells such as, but not limited to, osteoblasts,osteoprogenitor cells, stem cells, bone marrow cells, fibroblasticcells, dural cells, periosteal cells, pericytes, and/or muscle cells.

In various embodiments, the use of ISN-1 and LNell-1 can be combined. Itis expected that ISN-1 and LNell-1 can work in synergy and thecombination provides improvements to the existing approaches. In variousembodiments, the isoform Nell-1 can be SNell-1.

Composition

In various embodiments, this invention provides a composition useful forfacilitating bone formation or regeneration. The composition comprisesan ISN-1 peptide, an ISN-1 related agent, or a combination thereof.

The composition can be a pharmaceutical composition which comprises apharmaceutically acceptable carrier. The pharmaceutically acceptablecarrier can be a carrier for a mode of delivery of oral administration,topical administration, in situ implant, intravenous administration,parenteral administration, local administration, intra-arterialinjection, injection into a fracture site, and delivery in abiodegradable matrix. In the various embodiments, the composition canfurther comprise LNell-1 protein, a related agent, or a combinationthereof.

The pharmaceutical composition can be formulated into a formulationsuitable for a mode of delivery selected from percutaneous injectionthrough intact skin to a site, direct injection through a surgicallyopened site or a trauma site, surgical implantation, extravasculardelivery, extravascular injection, extravascular catheter basedinjection, intravascular delivery, intravascular injection,intravascular catheter based injections, intravenous delivery,intravenous injection, intravenous catheter based injections,intraarterial delivery, intraarterial injection, intraarterial catheterbased injections, intrathecal delivery, intrathecal injection,intrathecal catheter based injections, intraosseous delivery,intraosseous injection, catheter based injections, intracartilaginousdelivery, intracartilaginous injection, intracartilaginous catheterbased injections, intravesical delivery, intravesical injection,intravesical catheter based injection, delivery via a mechanical pumpwith a percutaneous or implantable catheter, catheter based delivery toan area or organ in the body, or delivery via expanded dispersionthrough a device that increases tissue penetration or wider tissuedistribution.

In various embodiments, the use of ISN-1 and LNell-1 can be combined.ISN-1 and LNell-1 can be used in the same fashion or substantially thesame fashion. It is expected that ISN-1 and LNell-1 can work in synergyand the combination provides improvements to the existing approaches. Invarious embodiments, the isoform Nell-1 can be SNell-1.

Bone Graft

In various embodiments, the composition can be a bone graft material. Invarious embodiments, this invention provides a bone graft material forenhancing bone formation in the animal in which it is implanted.

In some embodiments, the bone graft material contains a biocompatiblematrix and an ISN-1 protein, a related agent, or a combination thereof.In some embodiments, the graft material can be resorbable orbiodegradeable or biostable. In some embodiments, the graft material canbe synthetic or naturally occurring (e.g., allograft). The matrix caninclude a biodegradable polymer or a biostable polymer and can beimpregnated with an ISN-1 protein, a related agent, and/or a cellexpressing an ISN-1 protein or a related agent. The biocompatible matrixcan comprise collagen. The biocompatible matrix can comprise a bioglassor a bioceramics. The biocompatible matrix can comprise a cell adhesionmolecule.

In some embodiments, the ISN-1 protein is produced by a cell within thematrix expressing the ISN-1 protein or a related agent, which areexogenous. In some embodiments, the ISN-1 protein is provided viapharmaceutical composition. In the aforementioned embodiments, the bonegraft material can further comprise LNell-1 protein, a related agent, ora combination thereof.

An exemplary bone graft material comprises a collagen conjugatecontaining (e.g. from about 0.001 to about 99.999 weight percent)collagen having dispersed substantially uniformly therein; and (e.g.about 99.999 to about 0.001 weight percent) an ISN-1 protein, a relatedagent, and/or a cell expressing an ISN-1 protein or a related agent. Insome embodiments, the graft material includes collagen and/ordemineralized or non-demineralized bone fragments in addition to theISN-1 protein or cells expressing an ISN-1 protein.

Cells expressing or over expressing ISN-1 can be incorporated into suchbone graft materials or ISN-1 polypeptides can be incorporated into suchbone graft materials. These graft materials can be used in the treatmentof fractures or to facilitate the replacement/healing of prostheses orbone transplants.

In various embodiments, the use of ISN-1 and LNell-1 can be combined. Itis expected that ISN-1 and LNell-1 can work in synergy and thecombination provides improvements to the existing approaches. In variousembodiments, the isoform Nell-1 can be SNell-1.

Method of Making Isoform Nell-1 Peptide

The expression and purification of Nell-1 peptide has been described inU.S. Pat. No. 7,544,486 and U.S. application Ser. No. 11/601,529, theteachings of which are incorporated by reference herein.

This invention provides methods for the expression and purification ofthe isoform Nell-1. In various embodiments, this invention providesnucleic acid constructs expressing ISN-1 and cells expressing ISN-1peptides which may be useful in producing quantities of ISN-1 peptides.In some embodiments, the nucleic acid constructs expressing ISN-1 mayfurther include nucleic acid sequences encoding signal peptides whichmay facilitate the protein trafficking and post production modificationof the ISN-1 in the host cell. In some embodiments, the signal peptidemay facilitate the secretion of the peptide from the host cell.

In various embodiments, this invention provides a method of expressing afunctional ISN-1 peptide in a cell, said method comprising: providing anucleic acid construct including at least a nucleic acid encoding atleast an ISN-1 peptide in frame with a nucleic acid encoding a secretorysignal peptide; transfecting a cell with said nucleic acid construct;culturing said cell under conditions that permit expression of the ISN-1peptide; optionally collecting ISN-1 peptide secreted from the cellline; optionally substantially purifying the ISN-1 peptide; andoptionally testing the activity of the ISN-1 peptide to induce boneformation.

In various embodiments, this invention provides a nucleic acid constructfor expressing an ISN-1 peptide in a cell, said nucleic acid constructcomprising at least a nucleic acid encoding at least an ISN-1 peptide inframe with a nucleic acid encoding a secretory signal peptide.

In various embodiments, this invention provides a cell line forexpressing a functional ISN-1 peptide, said cell line including anucleic acid construct comprising at least a nucleic acid encoding atleast an ISN-1 peptide in frame with a nucleic acid encoding a secretorysignal peptide.

In some embodiments, the secretory signal peptide is an insect secretorypeptide. In some embodiments, the secretory signal peptide is a Nellpeptide signal sequence. In some embodiments, the secretory signalpeptide is selected from the group consisting of a melittin signalsequence, a drosphila immunoglobulin-binding protein signal sequence, anequine interferon-gamma (elFN-gamma) signal peptide, a snakephospholipase A2 inhibitor signal peptide, a human lysozyme signalpeptide, and a chicken lyzozyme signal peptide.

In the aforementioned embodiments, the cell can be a mammalian cell oran insect cell. Said insect cell can be a high five cell. Said mammaliancell can be a COS7 cell. In the aforementioned embodiments, thesecretory signal peptide can be a Nell peptide signal sequence. In theaforementioned embodiments, the ISN-1 peptide can be SNell-1.

In various embodiments, the isoform Nell-1 can be SNell-1.

U.S. Pat. Nos. 7,052,856 and 7,544,486, U.S. application Ser. Nos.11/392,294, 11/594,510, 11/601,529, 11/713,366, 11/884,525, and11/973,831 describe Nell-1 peptide, compositions thereof, its expressionand purification, and its use in bone fracture repair includingfacilitating bone formation and increasing bone mineralization, treatingosteoporosis, and inducing cartilage formation and regeneration. Thesepatents or applications provide the state of art which contributes tothe enablement to various aspects of the present invention.

Embodiments

The method of bone formation and repair generally involves increasingISN-1 protein concentration in a bone progenitor cell or contacting acell (e.g. a bone progenitor cell) with an ISN-1 polypeptide or with avector encoding an ISN-1 polypeptide.

This can be accomplished by transforming a bone precursor cell so thatit expresses elevated levels of endogenous ISN-1 or so that it expressesISN-1 from an exogenous transfected ISN-1 nucleic acid.

This also can be accomplished by contacting bone precursor cells at ornear the bone, bone fracture site, or cartilage disease site with anISN-1 protein or a composition thereof or local administration of anISN-1 protein or a composition thereof.

A) Transformation of Cells to Increase ISN-1 Production.

In a more preferred embodiment, the ISN-1 gene expressing nucleic acids(e.g. cDNA(s)) can be cloned into gene therapy vectors that arecompetent to transfect cells (such as human or other mammalian cells) invitro and/or in vivo. The methods and procedures of such cloning andcell transfection are described in U.S. application Ser. No. 09/412,297,filed on Oct. 5, 1999, the teachings of which are incorporated herein intheir entirety by reference.

B) Administration of Exogenously Produced ISN-1

1) Delivery of ISN-1 to Target Cells

ISN-1 proteins or related agents can be prepared for intravenous,parenteral, topical, oral, or local administration (e.g. by aerosol ortransdermally). Particularly preferred modes of administration includeintra-arterial injection, injection into fracture sites or delivery in abiodegradable matrix. The ISN-1 proteins agents can be combined with apharmaceutically acceptable carrier, which can be referred to as carrieror excipient, to form a pharmacological composition.

Various pharmaceutically suitable formulations, carriers, otheradditives, and administering routes are described in U.S. applicationSer. Nos. 11/392,294, 11/713,366, 11/884,525, and 11/973,831, which areincorporated herein by reference.

2) Bone Graft Materials

ISN-1 protein can be applied directly to a bone or bone fracture site.This can be accomplished during surgery (e.g. when setting complexfractures, when reconstructing bone, when performing bone transplants,etc.) or can be accomplished by direct injection.

In certain preferred embodiments, particularly where bone reconstructionor repair is performed surgically, it is desired to administer the ISN-1protein using a sustained delivery “vehicle”. Sustained deliveryvehicles include, but are not limited to biodegradable deliveryvehicles. Biodegradable delivery vehicles are preferably porous. Variousdelivery vehicles are described in U.S. application Ser. Nos.11/392,294, 11/713,366, 11/884,525, and 11/973,831, which areincorporated herein by reference.

Other delivery vehicles include, but are not limited to bone graftmaterials. Bone graft materials can be derived from natural materials(e.g. transplanted bone or bone fragments), synthetic materials (e.g.various polymers and/or ceramics) or combinations of both. Bone graftmaterials are typically utilized to fill voids or otherwise replace lostbone material. Such graft materials are also often provided ascomponents of prosthetic devices (e.g. bone replacements or supports) tofacilitate tight bonding/annealing of the prosthetic with the livingbone. The bone graft material can include a biodegradable polymer or abiostable polymer.

Bone grafts using bioactive glasses and calcium phosphates, collagen,mixtures and the like have good biocompatibility and give rise to bonetissue formation and incorporation in some cases. Various bone graftmaterials are described in U.S. application Ser. Nos. 11/392,294,11/713,366, 11/884,525, and 11/973,831, which are incorporated herein byreference.

The bone graft material can further include bone morphogenic proteins(BMP) or other bioactive agents such as cell adhesion molecules.Particularly suitable graft materials include, for example, isolatedmineralized cancellous bone sections, powders or granules of mineralizedbone, demineralized cancellous bone sections, powders or granules ofdemineralized bone, guanidine-HCl extracted demineralized bone matrix,sintered cortical or cancellous bone, coralline hydroxyapatite sold byInterpore under the trade name Interpore 500, and granular ceramics suchas that incorporated into the bone graft substitute Collagraft sold byZimmer, or filamentous sponges such as those made from collagen byOrquest.

In various embodiments, the ISN-1 proteins, BMP, or other bioactiveagent can be bound to the substrate of the bone graft materials.

3) ISN-1 for Osteoporosis

In some embodiments, the ISN-1 protein described herein can be used totreat, prevent, or ameliorate osteoporosis. In this embodiment, theISN-1 peptide can be administered to a site of osteoporosis.Subsequently, a physical force such as a vibration or ultrasound can beapplied to the site of administration to disperse the ISN-1 peptide. Insome embodiments, the ISN-1 peptide can be administered to the site ofosteoporosis by the acts of (a) making an incision in a tissue (bone)and (b) delivering to the tissue through the incision the ISN-1 peptide.In some embodiments, the Nell-1 peptide can be in a pharmaceuticallyacceptable carrier for sustained delivery.

4) ISN-1 for Cartilage Regeneration

In the present invention, the isoform Nell-1 peptide can be used totreat, prevent, or ameliorate cartilage degeneration. In one embodiment,the ISN-1 peptide can be administered to a site of fibrocartilagedisease such as spinal disc disease with or without a pharmaceuticallyacceptable carrier, with or without other devices (e.g., disc nucleusreplacement device, allograft device, or cells) or biological factors(e.g., LIM-1 protein). In another embodiment, the ISN-1 peptide can beadministered to a site of fibrocartilage disease such as meniscus, withor without a pharmaceutically acceptable carrier, with or without otherdevices (e.g., meniscus allograft or meniscus scaffold or prosthesis, orcells) or biological factors. In another embodiment, the SNell-1 peptidecan be administered to a site of hyaline cartilage disease such as kneearticular cartilage, with or without a pharmaceutically acceptablecarrier, with or without other devices (e.g., cartilage allograft orcartilage scaffold or prosthesis) or biological factors. In anotherembodiment, the ISN-1 peptide can be administered to another site ofhyaline cartilage disease such as tracheal cartilage (e.g.,tracheomalacia), with or without a pharmaceutically acceptable carrier,with or without other devices (e.g., cartilage allograft or cartilagescaffold or prosthesis) or biological factors.

In other embodiments, the ISN-1 peptide can be administered to a site ofelastic cartilage disease such as auricular or epiglottis with orwithout a pharmaceutically acceptable carrier, with or without otherdevices (e.g., cells) or biological factors.

A composition described herein can be formulated into formulationssuitable for any suitable mode of administration/delivery to a mammaliansubject (e.g., a human being). An ordinary artisan with the teachingsabove can formulate the composition described here into any desirableformulation by using, e.g., an appropriate carrier with an appropriateamount of an ISN-1 peptide or a related agent defined above.

Some examples of delivering the composition can be, e.g., percutaneousinjection through intact skin to various sites, or direct injectionthrough nonintact skin (e.g., surgically opened sites or trauma sites).In some embodiments, the delivery can be surgical implantation of acomposition described herein. In some embodiments, the delivery can beone of extravascular delivery, injection or catheter based injections;intravascular delivery, injection or catheter based injections;intravenous delivery, injection or catheter based injections;intraarterial delivery, injection or catheter based injections;intrathecal delivery, injection or catheter based injections;intraosseous delivery, injection or catheter based injections;intracartilaginous delivery, injection or catheter based injections; orintravesical delivery, injection or catheter based injections.

In some embodiments, a delivery of composition described herein to amammalian subject can be delivery via mechanical pumps with percutaneousor implantable catheters. In some embodiments, a delivery of compositiondescribed herein to a mammalian subject can be catheter based deliveryto any area/organ in the body.

In some embodiments, a delivery of composition described herein to amammalian subject can be delivery via expanded dispersion throughvarious devices promoting increased tissue penetration or wider tissuedistribution (e.g., ultrasound, iontophoresis, heat, pressure, etc.)

EXAMPLES

The following example illustrates, but not to limit the claimedinvention.

1. SNell-1

An optional open reading frame in exon 7 within 240 amino acids from thefirst open reading frame (ORF) that lacks the TSP1-N like domain insilico search was identified. The ATG potential translational start sitefor SNell-1 site is located within exon 7 of LNell-1 and the promotersequences for SNell-1 are within intron 2 of LNell-1. Based on 5′ RACEand sequencing results, the existence of the predicted short form wasfurther demonstrated. Independent work by Database of TranscriptionalStarting Sites (http://dbtss.hgc.jp) using 5′ Oligo-Capping method alsoidentified a clone with the same alternative form for SNell-1.

SNell-1 5′ primer was designed based on its 5′ UTR sequence. Whentransfected with Osx, the PCR product using N-terminal primers (specificfor LNell-1) was significantly downregulated (coinciding with thepromoter result), while the PCR product using SNell-1 specific primerswas significantly elevated above control (FIG. 2). In contrast, Runx2transfection upregulated both LNell-1 and SNell-1 transcripts.

LNell-1 contains 810 aa with a molecular weight of 89.5 kD (˜120 kDafter post-translational modification); the predicted size for SNell-1is 570 aa with a molecular weight of 62.5 kD.

To further verify the existence of this smaller isoform, the media fromOsx transfected NMCCs was collected. It was shown that Osx downregulatedLNell-1 and increased the expression of an ˜70 kD SNell-1 protein thatis consistent with the predicted nonglycosylated weight of 62.5 kD (FIG.2). Similar results were obtained using Saos2 cells.

2. Functional Role of SNell-1 in Skeletal Development

A Nell-1 gene-trapped ES cell line was used to generate general andtissue specific [Col1α1-Cre-(osteoblastic) and Col2α1-Cre(chondrogenic)] Nell-1 knockouts. LNell-1 and SNell-1 overexpressingmice were generated. In addition to comprehensive morphological andhistological examination, levels of Runx2 and Osx expression werefurther examined on the different Nell-1 expression backgrounds byimmunohistochemistry.

Given the important roles of Runx2 and Osx during skeletal development,SNell-1's effect on Runx2 and Osx expression were studied. Becausephosphorylation status can affect Runx2 and Osx activity and becauseLNell-1 has been demonstrated to increase Runx2 phosphorylation andactivity, SNell-1's effects on Runx2 and Osx phosphorylation status andactivity were studied.

SNell-1 is normally expressed by late-stage osteoblasts. ExcessiveSNell-1 may induce more significant cellular apoptosis and greaterinhibition of cell proliferation than LNell-1, with accelerated boneformation relative to WT mice, but decreased total bone formationrelative to LNell-1 overexpression mice.

In terms of chondrogenesis, SNell-1 (which Runx2 and Osx upregulates andwhich reciprocally upregulates Runx2 and Osx mRNA) promotes earlychondrocyte differentiation, with possible inhibitory effects onterminal chondrocyte differentiation.

The differential effects of LNell-1 vs. SNell-1 in Saos-2 cells wereexamined. Saos-2 transfected with pcDNA3.1-SNell-1 demonstratedincreased Runx2, Osx, and Oc expression by day 6 of culture. Incontrast, LNell-1 transfected Saos-2 cells demonstrated no change inRunx2 transcription (consistent with our previous data in MC3T3-E1),transiently increased Oc expression, and marked decrease in Osxexpression (FIG. 3).

FIG. 3 shows the effects of LNell-1 and SNell-1 on Runx2, Osx, Ocexpression and mineralization. FIG. 3(A) shows that Saos2 cells weretransfected with pcDNA3.1 (Control), pcDNA3.1-LNell-1 or -SNell-1 for 24hours and then exposed to osteogenic differentiation medium. mRNAexpression of Runx2, Osx and Oc was analyzed by real-time PCR. *p<0.05,**p<0.01 compared to data of day 1 within same group. #p<0.05, ##p<0.01compared to control group within same time point. FIG. 3(B) showsLentiviral transfection. Both LNELL-1 and SNell-1 show significantmineralization compared to control. #p<0.05.

SNell-1 was expressed during skeletal growth (FIG. 4) and formed bonewhen applied to calvarial defects (FIG. 5). FIG. 4 shows LNell-1 andSNell-1 protein express patterns in mice heads. LNell-1 is known to behighly expressed in brain tissues and is consistently expressed duringand after gestation while SNell-1 is predominantly expressedpostnatally.

FIG. 5 shows Calvarial defect healing with SNell-1. SNell-1 lentivirus(5M virus particles per site) embedded in collagen coated PLGA scaffoldswere implanted onto 3 mm calvarial defect in athymic rats. Live μCT weretaken at 2 weeks and high resolution μCT taken at 4 weeks. BV/TV andbone surface density both showed that SNell-1 induced significantly morebone formation (*p<0.05).

These data demonstrate that SNell-1 exhibits potent osteoinductivecapability. It was found that isoform Nell-1 peptides are required fornormal skeletal development and that isoform Nell-1 peptides are keycomponents in the Runx2 and Osx regulatory network controllingosteoblastogenesis and terminal chondrocyte differentiation.

LNell-1 may be involved in earlier-stage and SNell-1 in later-stageosteoblastogenesis, and the converse for chondroblastogenesis (i.e.,LNell-1 may be involved in later-stage; SNell-1 in earlier-stage).LNell-1 (upregulated by Runx2 and downregulated by Osx) may promoteearlier stage osteoblast differentiation and less mature bone formation,while SNell-1 (upregulated by Osx and Runx2) may promote later stageosteoblast differentiation and more mature bone formation.

Conversely, for chondrocytes, Runx2 upregulated LNell-1 may promoteterminal chondrocyte maturation (given the known roles of Runx2 inpromoting chondrocyte hypertrophy) and Osx/Runx2 upregulated SNell-1 mayhave minimal or perhaps even inhibitory effects on terminal chondrocytedifferentiation (given the known inhibitory effects of Osx onchondrocyte hypertrophy).

These data suggest that SNell-1 can lead to safer and more effectiveosteoinductive therapies.

3. Isoform Nell-1's Effects on Runx2 and Osx Phosphorylation andActivity

Coordinated regulation of Runx2 and Osx activity are crucial for boneformation. Studies by the present inventors suggest that LNell-1 andSNell-1 are important, not only as target genes for carrying out Runx2and Osx functions, but that LNell-1 and SNell-1 are important formodulating Runx2 and Osx expression and activity during celldifferentiation. Understanding of isoform Nell-1's function andmechanism will lead to improved and safer therapeutic approaches toconditions related to bone formation.

The effects of isoform Nell-1 on Runx2 phosphorylation status wereexamined and correlated with Runx2 activity. Runx2 activity wasquantitated physiologically by osteoblastic marker expression, anddirectly by luciferase reporter systems that serve as direct readouts ofRunx2 activity.

Because LNell-1 increases Runx2 phosphorylation through involvement ofmitogen-activated kinase signaling (MAPK) cascades, and contains aconserved TSP1-N/LG-like domain that may interact with cell-surfaceintegrins to activate MAPK pathways, the relative levels of MAPK andfocal adhesion kinase (FAK) activation by LNell-1 and SNell-1 were alsoexamined.

LNell-1 and SNell-1 exerted different effects on Runx2 and Osxphosphorylation and activity with correspondingly different effects onosteogenic and chondrogenic differentiation.

The effects of single or combined Nell-1 isoforms on Runx2 and Osxphosphorylation status and corresponding bioactivities were alsoexamined. Combined LNell-1 and SNell-1 can synergistically increaseRunx2 activity and osteoblast differentiation and therefore provide acombined therapy.

4. Isoform Nell-1' Effects on Bone Formation In Vivo

The osteoinductivity of LNell-1 and SNell-1 in a calvarial defect modeland a bone marrow stem cell (BMSC) implant model were examined.

Both Runx2 and Osx are known to promote osteoblast lineage commitmentand maturation. However, while Runx2 promotes terminal chondrocytedifferentiation, Osx inhibits this process. Osx-induced SNell-1 willpreferentially promote an intramembranous ossification-like process ofdirect osteogenesis (i.e., mesenchymal stem cells differentiating intoosteoblasts), while LNell-1, which is specifically upregulated by Runx2and downregulated by Osx, can preferentially promote a more endochondralossification-like process of step-wise osteogenesis (e.g., mesenchymalstem cells differentiating into chondrocytes with formation of calcifiedmatrix, followed by vascular invasion and influx of new mesenchymal stemcells that then differentiate into osteoblasts).

It is expected that combined LNell-1 and SNell-1 therapies are moreefficacious for bone growth at lower doses—allowing further optimizationof Nell-1 safety and efficacy. The BMSC study will also determine how touse the two isoform Nell-1 peptides as novel molecular tools to controlbone vs. cartilage formation in healing bone fractures so thatdevelopment of excessive cartilage in the fracture callus can beminimized and direct, or step-wise, bone formation maximized.

To determine the relative osteoinductive and chondroinductive propertiesof LNell-1 vs. SNell-1 vs. combination LNell-1/SNell-1 in complex invivo environments, two models were used—an established calvarial defectmodel of intramembranous bone regeneration that does not form chondroidbone and a BMSC implantation model that more closely resemblesendochondral bone regeneration with osteochondral bone formation.Quantitative and qualitative bone formation was evaluated at grossmorphologic, histologic, and molecular levels.

The osteoinductive properties of rhLNell-1 vs. rhSNell-1 in anintramembranous ossification model were studied. Previous data indicatesthat LNell-1 can accelerate both chondrogenesis and osteogenesis. Incontrast, SNell-1, unlike LNell-1, can primarily promoteosteoblastogenesis with minimal (or perhaps even inhibitory effects) onterminal chondrocyte differentiation. It is expected that SNell-1 willinduce faster and more mature bone which manifests as increased bonevolume/density, increased or earlier expression of osteoblastic markergenes, and/or more mature bone trabecular patterns on histology.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1. A method of increasing bone formation or regeneration at or near abone fracture site in a subject in need thereof, the method comprisingincreasing the concentration of an ISN-1 gene product at the bonefracture site.
 2. The method of claim 1, wherein the increasingconcentration of an ISN-1 gene product comprises administering to thebone fracture site with a pharmaceutical composition comprising theISN-1, a related agent, or a combination thereof.
 3. The method of claim2, wherein the pharmaceutical composition further comprising LNell-1protein, a related agent, or a combination thereof.
 4. A method oftreating, preventing or ameliorating osteoporosis by administering to abone tissue at a pre-selected site an effective amount of an ISN-1, arelated agent, or a combination thereof.
 5. The method of claim 4,further comprising applying to the pre-selected site a physical force todisperse the ISN-1.
 6. The method of claim 4, wherein the administeringstep comprises: making an incision in the bone tissue at thepre-selected site, and delivering to the bone tissue at the pre-selectedsite via the incision.
 7. The method of claim 4, wherein the physicalforce is ultrasound.
 8. The method of claim 4, wherein the ISN-1 isformulated into a formulation suitable for a mode of delivery selectedfrom percutaneous injection through intact skin to a site, directinjection through a surgically opened site or a trauma site, surgicalimplantation, extravascular delivery, extravascular injection,extravascular catheter based injection, intravascular delivery,intravascular injection, intravascular catheter based injections,intravenous delivery, intravenous injection, intravenous catheter basedinjections, intraarterial delivery, intraarterial injection,intraarterial catheter based injections, intrathecal delivery,intrathecal injection, intrathecal catheter based injections,intraosseous delivery, intraosseous injection, catheter basedinjections, intracartilaginous delivery, intracartilaginous injection,intracartilaginous catheter based injections, intravesical delivery,intravesical injection, intravesical catheter based injection, deliveryvia a mechanical pump with a percutaneous or implantable catheter,catheter based delivery to an area or organ in the body, or delivery viaexpanded dispersion through a device that increases tissue penetrationor wider tissue distribution.
 9. The method of claim 8, wherein thedevice provides ultrasound, iontophoresis, heat or pressure.
 10. Themethod of claim 1 wherein ISN-1 consists of the amino acid sequenceaccording to SEQ ID NO:
 1. 11. The method of claim 1 wherein the ISN-1further comprises at least one posttranslational chemical modification.12. The method of claim 11 wherein the posttranslational chemicalmodifications are selected from the list consisting of short hydrocarbongroup addition; mono(ethylene glycol) addition; oligo(ethylene glycol)addition; and poly(ethylene glycol) addition.
 13. The method of claim 1wherein the ISN-1 is PEGylated.
 14. The method of claim 4 wherein ISN-1consists of the amino acid sequence according to SEQ ID NO:
 1. 15. Themethod of claim 14 wherein the posttranslational chemical modificationsare selected from the list consisting of short hydrocarbon groupaddition; mono(ethylene glycol) addition; oligo(ethylene glycol)addition; and poly(ethylene glycol) addition.
 16. The method of claim 4wherein the ISN-1 is PEGylated.
 17. A method of administration of anISN-1 agent, an ISN-1-related agent, or a combination thereof, fortreatment of osteoporosis to a patient in need thereof, comprising thesteps of: diagnosing a patient with osteoporosis; pre-selecting a tissuesite in need of osteoporosis amelioration; making in incision proximateto the tissue in need of osteoporosis amelioration; and delivering, viathe incision, a therapeutically effective amount of an ISN-1 agent, anISN-1-related agent, or a combination thereof.
 18. The method of claim17 wherein the ISN-1 agent or ISN-1-related agent or combination thereofis PEGylated.
 19. The method of claim 17 wherein the ISN-1 agentconsists of a protein having at least 90% homology with SEQ ID NO: 1.20. The method of claim 17 further comprising the step of dispersing theISN-1 agent, an ISN-1-related agent, or a combination thereof by meansof ultrasound.